This invention relates to nucleic acid molecules encoding human neuronal nicotinic acetylcholine receptor protein subunits, as well as the encoded proteins. In particular, human neuronal nicotinic acetylcholine receptor xcex1-subunit-encoding DNA and RNA, xcex1-subunit proteins, xcex2-subunit-encoding DNA and RNA, xcex2-subunit proteins, and combinations thereof are provided.
Ligand-gated ion channels provide a means for communication between cells of the central nervous system. These channels convert a signal (e.g, a chemical referred to as a neurotransmitter) that is released by one cell into an electrical signal that propagates along a target cell membrane. A variety of neurotransmitters and neurotransmitter receptors exist in the central and peripheral nervous systems. Five families of ligand-gated receptors, including the nicotinic acetylcholine receptors (nAChRs) of neuromuscular and neuronal origins, have been identified (Stroud et al. 1990 Biochemistry 29:11009-11023). There is, however, little understanding of the manner in which the variety of receptors generates different responses to neurotransmitters or to other modulating ligands in different regions of the nervous system.
The nicotinic acetylcholine receptors (nAChRs) are multisubunit proteins of neuromuscular and neuronal origins. These receptors form ligand-gated ion channels that mediate synaptic transmission between nerve and muscle and between neurons upon interaction with the neurotransmitter acetylcholine (ACh). Since various neuronal nicotinic acetylcholine receptor (nAChR) subunits exist, a variety of nAChR compositions (i.e., combinations of subunits) exist. The different nAChR compositions exhibit different specificities for various ligands and are thereby pharmacologically distinguishable. Thus, the nicotinic acetylcholine receptors expressed at the vertebrate neuromuscular junction, in vertebrate sympathetic ganglia and in the vertebrate central nervous system have been distinguished on the basis of the effects of various ligands that bind to different nAChR compositions. For example, the elapid xcex1-neurotoxins that block activation of nicotinic acetylcholine receptors at the neuromuscular junction do not block activation of some neuronal nicotinic acetylcholine receptors that are expressed on several different neuron-derived cell lines.
Muscle nAChR is a glycoprotein composed of five subunits with the stoichimetry (xcex1)2xcex2(xcex3 or xcex5)xcex4. Each of the subunits has a mass of about 50-60 kilodaltons (kd) and is encoded by a different gene. The (xcex1)2xcex2(xcex3 or xcex5)xcex4 complex forms functional receptors containing two ligand binding sites and a ligand-gated transmembrane channel. Upon interaction with a cholinergic agonist, muscle nicotinic nAChRs conduct sodium ions. The influx of sodium ions rapidly short-circuits the normal ionic gradient maintained across the plasma membrane, thereby depolarizing the membrane. By reducing the potential difference across the membrane, a chemical signal is transduced into an electrical signal at the neuromuscular junction that induces muscle contraction.
Functional muscle nicotinic acetylcholine receptors have been formed with xcex1xcex2xcex4xcex3 subunits, xcex1xcex2xcex3 subunits, xcex1xcex2xcex4 subunits, xcex1xcex4xcex3 subunits, but not only with one subunit (see, eg., Kurosaki et al. (1987) FEBS Lett. 214 253-258; Comacho et al. (1993) J. Neuroscience 13:605-613). In contrast, functional neuronal nAChRs can be formed from a subunits alone or combinations of xcex1 and xcex2 subunits. The larger a subunit is generally believed to be a ACh-binding subunit and the lower molecular weight xcex2 subunit is generally believed to be the structural subunit, although it has not been definitely demonstrated that the xcex2 subunit does not have the ability to bind ACh or participate in the formation of the ACh binding site. Each of the subunits which participate in the formation of a functional ion channel are, to the extent they contribute to the structure of the resulting channel, xe2x80x9cstructuralxe2x80x9d subunits, regardless of their ability (or inability) to bind ACh. Neuronal nAChRs, which are also ligand-gated ion channels, are expressed in ganglia of the autonomic nervous system and in the central nervous system (where they mediate signal transmission), and in pre- and extra-synaptic locations (where they modulate neurotransmission and may have additional functions; Wonnacott et al. (1990) In: progress in Brain Research, A. Nordberg et al., Eds., Elsevier, Amsterdam) 157-163.
DNA encoding nAChRs has been isolated from several sources. Based on the information available from such work, it has been evident for some time that nAChRs expressed in muscle, in autonomic ganglia, and in the central nervous system are functionally diverse. This functional diversity could be due, at least in part, to the large number of different nAChR subunits which exist. There is an incomplete understanding, however, of how (and which) nAChR subunits combine to generate unique nAChR subtypes, particularly in neuronal cells. Indeed, there is evidence that only certain nAChR subtypes may be involved in disease such as Alzheimer""s disease. Moreover, it is not clear whether nAChRs from analogous tissues or cell types are similar across species.
Accordingly, there is a need for the isolation and characterization of DNAs encoding each human neuronal nAChR subunit, recombinant cells containing such subunits and receptors prepared therefrom. In order to study the function of human neuronal nAChRs and to obtain disease-specific pharmacologically active agents, there is also a need to obtain isolated (preferably purified) human neuronal nAChRs, and isolated (preferably purified) human neuronal nAChR subunits. In addition, there is also a need to develop assays to identify such pharmacologically active agents.
The availability of such nucleic acids, cells, receptor subunits and receptor compositions will eliminate the uncertainty of speculating as to human neuronal nAChR structure and function based on predictions drawn from non-human nAChR data, or human or non-human muscle or ganglia nAChR data.
Therefore, it is an object herein to isolate and characterize DNA encoding subunits of human neuronal nicotinic acetylcholine receptors. It is also an object herein to provide methods for recombinant production of human neuronal nicotinic acetylcholine receptor subunits. It is also an object herein to provide purified receptor subunits and to provide methods for screening compounds to identify compounds that modulate the activity of human neuronal nAChRs.
These and other objects will become apparent to those of skill in the art upon further study of the specification and claims.
Isolated nucleic acid molecules encoding human alpha (xcex1) and beta (xcex2) subunits of neuronal nAChRs are provided. In particular, isolated DNA and RNA molecules encoding human xcex16 and xcex23 subunits of neuronal nAChRs are provided. Messenger RNA and polypeptides encoded by the DNA are also provided.
Recombinant human neuronal nicotinic nAChR subunits, including xcex16 and xcex23 subunits, as well as methods for the production thereof are also provided. In addition, recombinant human neuronal nicotinic acetylcholine receptors containing at least one human neuronal nicotinic nAChR subunit are also provided, as well as methods for the production thereof. Also provided are recombinant neuronal nicotinic nAChRs that contain a mixture of one or more nAChR subunits encoded by a host cell, and one or more nAChR subunits encoded by heterologous DNA or RNA (i.e., DNA or RNA as described herein that has been introduced into the host cell), as well as methods for the production thereof.
Plasmids containing DNA encoding the above-described subunits are also provided. Recombinant cells containing the above-described DNA, mRNA or plasmids are also provided herein. Such cells are useful, for example, for replicating DNA, for producing human nAChR subunits and recombinant receptors, and for producing cells that express receptors containing one or more human subunits.
The DNA, RNA, vectors, receptor subunits, receptor subunit combinations and cells provided herein permit production of selected neuronal nicotinic nAChR receptor subtypes and specific combinations thereof, as well as antibodies to the receptor subunits. This provides a means to prepare synthetic or recombinant receptors and receptor subunits that are substantially free of contamination from many other receptor proteins whose presence can interfere with analysis of a single nAChR subtype. The availability of desired receptor subtypes makes it possible to observe the effect of a drug substance on a particular receptor subtype and to thereby perform initial in vitro screening of the drug substance in a test system that is specific for humans and specific for a human neuronal nicotinic nAChR subtype.
Also provided herein, are single-stranded probes containing portions of the DNA molecules described herein and antibodies that specifically bind to proteins encoded by the DNA. Also provided herein is an isolated nucleic acid molecule containing nucleotides 98-211 of SEQ ID NO: 15.
Proteins encoded by the DNA are also provided. The proteins may be prepared by expressing the DNA in a suitable prokaryotic or eukaryotic host cell and isolating the resulting protein.
Methods for identifying functional neuronal nicotinic acetylcholine receptor subunits and combinations thereof are also provided.
Assays for identifying compounds that modulate the activity of human nicotinic acetylcholine receptors are also provided. The ability to screen drug substances in vitro to determine the effect of the drug on specific receptor compositions should permit the development and screening of receptor subtype-specific or disease-specific drugs. Also, testing of single receptor subunits or specific receptor subtype combinations with a variety of potential agonists or antagonists provides additional information with respect to the function and activity of the individual subunits and should lead to the identification and design of compounds that are capable of very specific interaction with one or more of the receptor subunits or receptor subtypes. The resulting drugs should exhibit fewer unwanted side effects than drugs identified by screening with cells that express a variety of subtypes.
Further in relation to drug development and therapeutic treatment of various disease states, the availability of DNA and RNA encoding human neuronal nAChR subunits provides a means for identification of any alterations in such genes (e.g., mutations) that may correlate with the occurrence of certain disease states. In addition, the creation of animal models of such disease states becomes possible, by specifically introducing such mutations into synthetic DNA sequences which can then be introduced into laboratory animals or in vitro assay systems to determine the effects thereof.